Rapid, nongenomic effects of endogenous steroids (STs), such as regulation of local artery diameter, have been classically attributed to steroid receptors coupled to downstream signaling. Whether endogenous steroids that reach nM-?M levels in circulation (hormones) or locally (paracrine neurosteroids) can regulate artery diameter by steroid-ion channel protein direct interactions remains largely unknown. Ion channel function often results from concerted actions of channel-forming and regulatory subunits. Smooth muscle (SM)-abundant ?1 subunits associate with channel-forming ? to increase the Ca2+-sensitivity of Ca2+/voltage-gated K+ channels (BK), which allows arterial SM BK to feedback on depolarization-driven Ca2+ influx and promote artery dilation. Aim 1 (lipid Molecular Pharmacology and vascular resistance Physiology) tests this central hypothesis: endogenous vasoactive STs regulate arterial diameter independently of ST receptors but through direct sensing of ST by specific sites in ? and ?1 subunits. Based on previous publications with cholesterol and lithocholate, as well as preliminary data, we will apply computational modeling and patch-clamp on recombinant channels expressed in SM cells from genetically engineered mice, to identify the molecular interactions between selected STs and specific BK amino acids, plus diameter evaluation in vessels of pathophysiological relevance: middle cerebral, basilar and mesenteric arteries, which express different levels of ?1. In parallel, Aim 2 (ion channel Biophysics and Medicinal Chemistry) combines pharmacophore optimization, compound prioritization+ functional assays with robotic patch-clamp, complex gating analysis, and computational docking to obtain two distinct classes of novel ?1-targeting nonsteroidal analogs (NSTAs) that present different structural basis of interaction with the protein and thus either activate ?1-containing BK or oppose this action. Finally, knowledge obtained with aims 1 and 2 will be integrated into Aim 3 (Translation Science), where we will demonstrate that the novel NSTAs that act as either agonists or antagonists on ?1-containing BK respectively behave either as endothelium-independent, artery dilators or oppose this action, with different arteries displaying differential sensitivity based on ?1 expression. We will further integrate the aims by showing that selective, new agents either counteract or synergize the vascular actions of endogenous STs. Finally, the efficacy of the new agents will be tested in a model of disease where the arterial tone is increased, i.e., the spontaneously hypertensive rat. Our focus on brain and peripheral arteries is most relevant to the therapy of human conditions that affect both vessels (e.g., hypertensive encephalopathy). ?1-based ligands that act independently of the endothelium will be particularly useful when endothelial function is impaired, such as with ageing or stroke. The combination of computational methods with electrophysiology on different point mutants as a strategy opposed to time-consuming structural methods, the use of high-throughput patch-clamp, and the feasibility of all experiments in our hands ensure the successful evaluation of several sites and ligands in the timeframe of an R01 award.